The use of single-stranded DNA or RNA probes to test for the presence of particular nucleic acids, and associated organisms and genetic features in biological materials is well known. Among areas in which such probes find usefulness include diagnostic testing of foods, blood and other biological specimens for infectious agents, diagnosis of genetic disorders and the presence of certain diseases such as cancers associated with genetic abnormalities. Non-isotopically labeled synthetic oligonucleotides are widely used in DNA sequencing, DNA hybridization assays, and more recently in amplification procedures, commonly known as polymerase chain reaction procedures described in U.S. Pat. Nos. 4,683,195 (issued Jul. 28, 1987 to Mullis et al) and 4,683,202 (issued Jul. 28, 1987 to Mullis).
The principle underlying the use of probes or primers is that under certain conditions, the probe or primer will hybridize by means of hydrogen bonding with a nucleic acid having complementary nucleotides. The hybridized product can then be suitably detected directly or after amplification procedures using appropriate reagents.
Early probes were labeled with radioisotopes such as .sup.32 P-labeled nucleotide triphosphates. However, they are unsuitable for many applications and are generally avoided due to safety and licensing considerations, and because of the natural decay of the label during storage.
Research has been continuing to find suitable labels for probes which do not have such disadvantages, as noted in EP-A-0 278 220 (published Aug. 17, 1988) and U.S. Pat. No. 4,780,405 (issued Oct. 25, 1988 to Kaiser et al). Enzyme labels have become the most generally used labels for labeled oligonucleotides, noted for example in EP-A-0 304 934 (published Mar. 1, 1989).
U.S. Ser. No. 103,978 (filed Oct. 2, 1987 by Levenson et al), now U.S. Pat. No. 4,962,029 (issued Oct. 9, 1990), describes the attachment of horseradish peroxidase to an oligonucleotide to form a covalent conjugate. In forming this conjugate, a mercapto-functionalized oligonucleotide is reacted with a maleimide-functionalized horseradish peroxidase. While this procedure represents an advance in the art, further improvements are desired to avoid undesirable side products, such as oxidation products of the mercapto-functionalized oligonucleotide, as well as the expensive and time-consuming preparative steps involved. Moreover, the thiol-functionalized oligonucleotide is unstable and has limited storage life. For maximum efficiency, it should be used as soon as it is prepared.
It would be highly advantageous, then, to have a method which would provide an enzyme-labeled oligonucleotide conjugate with improved yields and stability. It would also be desirable to be able to avoid side reactions of the critical oligonucleotide reagent.